Project Summary Cancer immunotherapy is now established as a major therapeutic modality. Significant progress with checkpoint inhibitor monoclonal antibodies demonstrates durable tumor regression in metastatic melanoma, lung cancer and other malignancies. One conspicuous limitation is the paucity of validated tumor antigens, which impedes clinical investigators in their pursuit of developing new cancer immunotherapies. Tumor encoded non-synonymous mutations may provide a new source of potential target antigens. T cells recognize tumor missense mutations as amino acid substituted peptides presented in the context of major histocompatibility molecules on the cancer cell surface, thus implicating missense mutations as a source of patient-specific neoantigens. We recently described the first-in-human study to use next generation sequencing technologies to identify and validate tumor missense mutations as neoantigens. Our study demonstrates that vaccination increases the breadth and diversity of neoantigen-specific T cells resulting in a broad repertoire of effector CD8+ T cells that uniquely discriminates mutated antigens from wild type peptides ensuring tumor specificity. This proposal aims to find a unique solution to the scarcity of tumor antigens by developing a neoantigen discovery pipeline that integrates genomic and proteomic technologies. In this proposal, a unique academic-industry partnership comprised of an established team of experts, in cancer genomics, cancer proteomics, human immunology, and clinical oncology, aim to solve the challenging problem of tumor neoantigen discovery. The long-term goal is to develop a robust pipeline using cutting-edge technologies merged with machine learning algorithms to supply tumor neoantigens for clinical investigation. The goal of this proposal will be addressed in the experiments of the following specific aims: 1) to identify the repertoire of melanoma expressed somatic mutations by next generation sequencing technologies; 2) to develop a novel proteomics platform to identify melanoma neoantigens presented by HLA class I molecules; 3) to validate neoantigen identification in a vaccination protocol. The proposed pipeline will deliver a transformative solution for tumor antigen identification, should be translatable to other ?immune responsive? malignancies and enable the nascent discipline of precision medicine to provide effective and safe immunotherapies for cancer patients.